Genome-wide association studies increasingly link human genetic variants to individual's response towards therapeutic agents. This increased knowledge greatly accelerates progress in personalized medicine. By identifying the genetic hotspots that are associated with drug efficacy and safety, pharmacogenomic (PGx) strategy allows for more individualized drug therapies based on the genetic make-up of patients. In turn, the individualized drug therapies may minimize side effects and improve outcomes (Daly, A K (2010) Nat Rev Genet, 11, 241-246). Already today, PGx information has been incorporated into a number of drug labels to assist clinicians to make therapeutic decisions.
One example is the PGx-guided warfarin dosing. Warfarin is the most widely described oral anticoagulant drug. Despite its effectiveness, warfarin is among the top 10 drugs with serious adverse event reports because of the narrow therapeutic index and the highly variable inter-individual dosing requirements. Therefore, it is important to monitor the anticoagulation status frequently with the International Normalized Ratio (INR), especially in the early period after the initiation of warfarin therapy. The lack of information available in identifying the appropriate initial dose usually leads to multiple dose adjustments and excesses risk of thromboembolic events or bleeding. In the last decade, PGx studies revealed correlations of the warfarin dose requirements and the presence of several genetic single-nucleotide polymorphisms (SNPs). The most significantly related SNPs include two coding variations in the cytochrome P450 enzyme CYP2C9 gene, CYP2C9*2 (rs 1799853) and CYP2C9*3 (rs1057910), and one variation in the vitamin K epoxide reductase complex 1 (VKORC1) gene, promoter SNP-1639G>A (rs9923231) (The International Warfarin Pharmacogenetics Consortium (2009) N Engl J Med, 360, 753-764). These SNPs may explain up to 35% of interpatient warfarin dose variability. In 2010, US FDA updated the labeling for warfarin with PGx-guided dosing ranges (Highlights of prescribing information. Coumadin (2010) http://packageinserts.bms.com/pi/pi_coumadin.pdf). The starting dose could be predicted by referring to a table containing stable maintenance doses observed in multiple patients having different combinations of CYP2C9 and VKORC1 variants. A dosing algorithm incorporating traditional clinical factors and patient genetic status is also available. Recently, a large-scale prospective study found that PGx-guided warfarin therapy reduced hospitalization rates for patients that just started a warfarin therapy by ˜30% (Epstein, R S et al. (2010) J Am Coll Cardiol, 22, 2804-2812). A randomized clinical trial, CoumaGen-II, provided further evidence for the clinical benefit of incorporating genotype knowledge into dosage selection (Anderson, J L et al. (2012) Circulation, 125, 1997-2005). As the value of warfarin genotyping is being supported by more and more clinical trials, increasing importance of the PGx results for clinical practice can be expected. Rapid and cost-effective genotype testing would greatly facilitate this process.
Currently available genotyping platforms include DNA microarray, real-time polymerase chain reaction (PCR), single-base extension, and high-resolution melting analysis (Kim, S and Misra, A (2007) Annu Rev Biomed Eng, 9, 289-320). Although these platforms are very useful in high-throughput studies, they are less cost-effective in on-demand clinical testing because of the expensive instruments and reagents involved (Joyce, H S (2011) Expert Opin Pharmacol, 12, 435-441). Four commercial assays have been approved by the FDA for warfarin-sensitivity genotyping, including Infinity Warfarin Assay (Autogenomics, Inc., Vista, Calif., USA), eSensor Warfarin Sensitivity Test (GenMark Diagnostics, Inc., Carlsbad, Calif., USA), eQ-PCR LC Warfarin Genotyping Kit (TrimGen, Sparks, Md., USA), and Verigene Warfarin Metabolism Nucleic Acid Test (Nanosphere, NorthBrook, Ill., USA). Some of these assays target point-of-care applications, but special instruments are required to conduct the testing.
Genotype analysis of highly heterogeneous specimens, such as tumor samples, is much more challenging than SNP genotyping because the somatic mutation detection has to be conducted in a large wild-type DNA background. For a heterozygous SNP sample, the MUT/WT allele ratio is 50%, while the ratio may be less than 10% for somatic mutation in a tumor specimen. A highly sensitive detection method is required to gauge the mutations. Usually, enrichment of the mutant sequences by allele-specific PCR or PCR clamping strategies are employed to achieve high sensitivity (Milbury, C A (2009) Clin Chem, 55, 632-640).
Recently, a new type of plasmonic nanoparticle (NP) probes, gold NPs functionalized with nonionic morpholino oligonucleotides (MORs) was prepared (Zu, Y et al. (2011) Small, 7, 306-310). The detection of DNA targets is based on the change of nanoprobe stability upon hybridization, i.e., the nanoprobes become more stable when bound to negatively charged DNA targets. The NP stability variation can be revealed simply by adding salt to the solution. The target-stabilized nanoprobes remain red in color, while the nanoprobes with no DNA attached would aggregate, leading to solution color change. The targets with similar sequences can be differentiated by various melting transition temperatures (Tm) of the target-probe hybrids (FIG. 1). The extremely sharp transition ensures the high detection specificity.
However, because of the low sensitivity of the nanoprobe-based method (detection limit ˜1 nM), genes cannot be analyzed directly. Prior to detection, amplification of the target sequence by PCR is necessary. In the present study, it has been found that the value of Tm is highly dependent on the target concentration and can be influenced significantly by the presence of salt in the assay solution. As PCR yield is usually unknown and different master solutions may contain various salt components, it is difficult to analyze PCR products by using a single set of nanoprobes and different, amplified template DNA molecules. Such detection method is described in WO 2011/087456.
Thus, despite major research efforts in recent years, no robust genetic testing platform has been develop yet that allows the determination of genotypes in combination with point-of-care use. Nonetheless, to accelerates progress in personalized medicine there is need in the art for such genetic testing platforms.